The invention relates to a method for regeneration of mentha plants from cell or tissue culture through organogenesis. More particularly, the invention relates to the use of menthol for the development of plant parts through organogenesis from calli in hormone free media.
The essential oil of Mentha arvensis Linn. var Piperascens is a well known source of the monoterpene xe2x80x98mentholxe2x80x99 used in the cosmetics, pharmaceutical, food, confectionery and liquor industries. The biosynthesis of menthol in the plant has a complex cascade fashion of genetic regulation during plant differentiation. Specific oil glands (trichomes) formed on the surface of the leaf and stem isolate these toxic monoterpenoid products to prevent the cellular damage. Monoterpenes are also known to be cytotoxic to plant tissues by acting through inhibition of respiration and photosynthesis, by drastically affecting the mitochondria, golgi bodies etc. and decreasing cell membrane permeability.
However, the present invention is to an experimental chance encounter while screening menthol tolerant clones of Mentha arvensis in tissue culture in presence of menthol in the medium. The applicants have developed a method for screening high menthol yielding genotypes in vitro in presence of menthol. While carrying out this large scale screening procedure, the applicants planned to subject the callus directly to menthol selection for selection tolerant clones. During this process, the applicants observed that menthol at toxic concentration induced the callus (transferred from other media) to form high frequency quality shoots better than those induced in presence of normally used growth hormones for shoot regeneration. This invention inspite of being simple may have far reaching consequences as a number of plants produce secondary metabolites causing end product toxicity and those upon testing may also be useful for organogenesis replacing the growth hormones.
In the present invention, the applicants observed that a lethal concentration of this toxic monoterpene (menthol) in medium induced Mentha arvensis callus (developed from other media), produced better quality shoots with much higher frequency than when induced by the normal growth hormones, contrary to the general observation that Mentha plants die when cultured in a medium containing menthol in toxic concentrations. There is no report of replacement of growth hormones by a monoterpene for inducing organogenesis from callus.
Therefore, the main object of the invention is to provide a method of regeneration of mentha plants from cell or tissue culture through organogenesis. Another object of the invention is to provide a method in which the use of menthol for the development of plant parts through organogenesis from calli in hormone free media is successfully carried out. Yet another object of the invention is to provide a novel and unique use of menthol as growth promoter, inducer of shoot regeneration and signal transducer, which can be provided in the hormone free culture medium for induction of shoots from undifferentiated mass of cells of Mentha arvensis. 
Accordingly, the present invention provide""s a method for the development of plants and plant parts through organogenesis from calli upon culturing in media comprising menthol in toxic concentrations.
Accordingly, the invention provides a novel and unique procedure of in vitro organogenesis from callus developed from any tissue culture media and then transferring it to medium free from growth hormones but containing menthol at a toxic concentration, wherein
a. the said callus is undifferentiated mass of cells,
b. the said toxic concentration is the concentration at which the regenerated shoots die which is 80 xcexcg mlxe2x88x921 or more in Mentha arvensis, 
c. the said menthol is produced from the essential oil of the plant of genus Mentha or synthesised chemically, and
d. the said growth hormones are but not limited to auxin, 1-napthalene acetic acid and cytokinin, benzyl aminopurine.
Another embodiment of the invention provides a method of producing organogenetic tissues from a plant of the genus Mentha, said method comprising the steps of:
(i) culturing an explant from a plant of genus Mentha in a initiation medium in the presence of 400 to 600 lux light at a temperature of 23-27xc2x0 C., with 16 hours of photoperiod, to obtain calli,
(ii) transferring the calli to a basal medium comprising minerals and vitamins to ensure development of shoots, and
(iii) culturing the shoots in a medium substantially free of growth hormones and containing menthol in a concentration of about 80 xcexcg mlxe2x88x921 to ensure root formation.
The preferred initiation medium employed, in the present invention is modified after MS medium, additionally comprising 0.2 xcexcg mlxe2x88x921 of auxins selected from naphthalene acetic acid or 1-NAA and 5 xcexcg mlxe2x88x921 of a cytokinin selected from BAP or benzyl aminopurine.
In another embodiment the invention provides a novel and unique use of menthol as growth promoter, inducer of shoot regeneration and signal transducer, which can be provided in the hormone free culture medium for induction of shoots from undifferentiated mass of cells of Mentha arvensis wherein
a. the menthol can replace the growth hormones for organogenesis wherein organogenesis comprises induction of differentiation from differentiated tissue like callus, and
b. the frequency of shoots generated is more in comparison to medium containing growth hormones.
The invention is described in detail hereafter with reference to the accompanying drawings following examples and FIG. 1, which are provided merely to illustrate the invention. Various modifications that may be apparent to one skilled in the art are deemed to fall within the scope of the present invention.